Photothermally responsive theranostic nanocomposites for near‐infrared light triggered drug release and enhanced synergism of photothermo‐chemotherapy for gastric cancer

Abstract Near‐infrared (NIR) photothermal therapy plays a critical role in the cancer treatment and diagnosis as a promising carcinoma treatment modalities nowadays. However, development of clinical application has been greatly limited due to the inefficient drug release and low tumor accumulation. Herein, we designed a NIR‐light triggered indocyanine green (ICG)‐based PCL core/P(MEO2MA‐b‐HMAM) shell nanocomposites (PPH@ICG) and evaluated their therapeutic effects in vitro and in vivo. The anticancer drug 5‐fluorouracil (5Fu) and the photothermal agent ICG were loaded into a thermo‐sensitive micelle (PPH@5Fu@ICG) by self‐assembly. The nanoparticles formed were characterized using transmission electron microscopy, dynamic light scattering, and fluorescence spectra. The thermo‐sensitive copolymer (PPH@5Fu@ICG) showed a great temperature‐controlled drug release response with lower critical solution temperature. In vitro cellular uptake and TEM imaging proved that PPH@5Fu@ICG nanoparticles can home into the lysosomal compartments under NIR. Moreover, in gastric tumor‐bearing nude mice, PPH@5Fu@ICG + NIR group exhibited excellent improvement in antitumor efficacy based on the NIR‐triggered thermo‐chemotherapy synergy, both in vitro and in vivo. In summary, the proposed strategy of synergistic photo‐hyperthermia chemotherapy effectively reduced the 5Fu dose, toxic or side effect, which could serve as a secure and efficient approach for cancer theranostics.


| INTRODUCTION
As one of the most common malignant cancer types of the digestive system, gastric cancer (GC) is the second leading cause of cancerrelated deaths worldwide. [1][2][3] Current therapies for GC, such as surgery, chemotherapy, and radiation therapy, are not yet satisfactory. 4 Thus, there is an urgent need for developing new therapeutic approaches for the treatment of GC. Polymeric nano drug carrier for GC chemotherapy plays a vital role in promoting the drug bioactivity and biocompatibility during the past decades, and have achieved certain therapeutic effects. 5 Recently, light triggered therapeutic approaches like photothermal therapy (PTT) for cancer treatment has received considerable attention due to its numerous superiority, such as minimal invasion, high spatiotemporal precision, and localized treatment. [6][7][8] It has been reported that near-infrared (NIR) photothermal agents, such as fluorescent dyes, gold nanomaterials, carbon nanotubes, and graphene oxide, could strongly absorb NIR laser and trigger temperature increase to induce the rapid drug release in thermo-responsive system. 9,10 The photothermo-chemotherapy employed NIR photosensitizer to generate hyperthermia (above 43 C) in target tumor regions by remotely controlled NIR-irradiation, which leaded to synergism by thermal ablation of cancer cells and enhanced antitumor drug release with significantly reduced toxic side effects, which are commonly produced by the single strategies like chemotherapy, radiotherapy, and surgery. 11,12 Indocyanine green (ICG) has been widely applied to biomedical imaging diagnosis and PTT with NIR fluorescence and 808 nm spectral absorption peak. 13,14 ICG has been approved for human use by Food and Drug Administration as a diagnostic cyanine NIR dye, which has ability of converting NIR light into heat. 15,16 Currently, NIR laser triggered hyperthermia has been developed to remotely control drug release in nanoparticles. NIR laser is an optimal choice for in vivo cancer therapy due to weaker tissue bioluminescence in 650-900 nm regions and less interference of biological background to fluorescence signal, which could provide strong tissue penetration for deep tissue imaging. [17][18][19] Controlled release of anticancer drugs could be released through NIR laser-triggered hyperthermia of thermo-responsive nano-carriers. 20 Notably, due to the different degree of the temperature sensitivity, hyperthermia can induce the death of cancer cells but not normal cells. 20 However, the applications of free ICG in NIR fluorescence imaging and PTT faced a series of restrictions owing to its drawbacks, including instability in aqueous solution and quick body clearance. 21,22 To overcome these challenges, various strategies have been developed to encapsulate ICG into nanocarriers, which provide enhanced prolonged circulation times and fluorescence stability in vivo. 23,24 The hyperthermia from ICG-micelles can lead to phase transition from the gel phase to the liquid crystalline phase, which promotes the drug release from micelles. Therefore, the ICG-loaded micelles would provide a new platform for cancer therapy to achieve a combinative effect of chemotherapy and photothermotherapy. 25 To achieve the NIR-triggered drug release and enhanced synergism of photothermo-chemotherapy, various stimuli-responsive nanocarriers respond to external stimulations (light, hyperthermia, magnetic field, and ultrasound) and internal stimulation (reduction/oxidation, pH, and enzyme) have been explored with perfect performances. 26,27 Thermoresponsive micelles have been deeply researched in oncotherapy and exhibit an enormous advantage in intelligent drug release among all the stimuli-responsive nanoparticles. 28 The thermo-responsive micelles realize the controlled drug release by a gel-to-liquid phase transition of thermo-responsive polymers in the region of their phase transition temperature, and the permeability of nanoparticles increases due to the instability of the shell, which facilitate rapid drug release. 29,30 Delivery efficiency of chemotherapeutic agent can be enhanced through optimizing the lower critical solution temperature (LCST) of the thermo-sensitive polymer and hyperthermia temperature of ICG.
Designing a new nano-platform that integrates a variety of imaging and treatment components for treatment of cancer therefore remains a challenge. In this study, 5-fluorouracil (5Fu) and ICG co-encapsulated thermo-sensitive micelles (PPH@5Fu@ICG) was prepared by selfassembly method (Scheme 1). This unique design could inhibit tumor growth through real-time tracking, NIR laser-driven drug release, and chemo/PTT. 5Fu, an agent for chemotherapy, is used as one of the standard chemotherapy regimens for GC. 31,32 The physiochemical characters were systematically investigated to explore the mechanism for drug release of PPH@5Fu@ICG. Fluorescence (FL) of ICG in PPH@5Fu@ICG was monitored to demonstrate subcellular localization, NIR laser-driven drug release, and metabolic distribution. The cytotoxic effects of PPH@5Fu@ICG combined controllable drug release, chemotherapy, and PTT were gradually evaluated in GC cells. Finally, the antitumor efficacy of PPH@5Fu@ICG in vivo through intratumoural injection was further appraised in comparison with single strategy.
Strikingly, the design is expected to use thermo-sensitive micelles to overcome the immune escape induced by chemotherapy, and reduce the side effects of chemotherapy through the photo-thermal response of ICG. The outcome of our work may be considered as a promising approach to integrate chemotherapy and PTT for GC therapy. Poly(ε-caprolactone) (PCL) was synthesized by ring-opening polymerization (ROP) of CL using dipentaerythritol as an initiator and Sn(Oct) 2 as a catalyst. 33 Briefly, CL (20.54 g, 180 mmol), dipentaerythritol (0.30 g, 1.18 mmol), and a catalytic amount of Sn(Oct) 2 were added to a flame-dried polymerization tube. The reaction tube was then flushed with argon and evacuated three times. After the stirring of the mixtures with a magnetic stirrer for 24 h at 115 C, the obtained crude product was dissolved in dichloromethane and then precipitated with iced methanol three times. Finally, the above samples were dried in a vacuum oven at 40 C to constant weight.

| Synthesis of PCL-Br
The PCL-Br was synthesized through esterification of 2-bromoisobutyryl bromide (BIBB), and the specific operations were as follows: 10 g of PCL (containing 3.46 mmol hydroxyl) was dissolved in 50 ml of dried chloroform under the protection of nitrogen atmosphere, and 1.5 g of triethylamine (14.82 mmol) was added. Then, the solution was cooled to 0 C in an ice bath, and BIBB (2.382 g, 10.36 mmol) dissolved in 20 ml of dried chloroform was added to the above system. The entire reaction system was stirred at room temperature for 24 h under argon protection, then filtered to remove insoluble salts. The filtrate was quenched three times with chloroform and deionized water (1:1, v/v). The organic phase was collected, and then solvents were removed by using a rotary evaporator.
Solid products were precipitated 2 times in iced methanol and dried in vacuum at room temperature to constant weight.

| NIR laser-triggered photothermal response in vitro
ICG (40, 60, and 80 μg/ml) and PPH@ICG (ICG: 40, 60, and 80 μg/ml) were dissolved in PBS and added into 6-well plate. The samples was irradiated by 808 nm NIR laser (1 W/cm 2 , 5 min). The PPH@ICG solution (ICG: 40 μg/ml) was irradiated by 808 nm NIR laser for 1 min, and then the irradiation was stopped to lower the temperature to the initial temperature. The experimental operation was carried out repeatedly for six times. The thermocouple thermometer is inserted into the liquid surface at room temperature and the temperature is recorded every 20 s. Thermographic camera images and temperatures were obtained by infrared imaging camera (Ti27, Fluke, USA).

| NIR triggered drug release in vitro
The encapsulation efficiency (EE) and drug loading capacity (LD) of PPH@5Fu@ICG were measured by a UV-vis spectrometer at 265 nm.

For in vitro experiments, human GC AGS cells were obtained from
Chinese Academy of Sciences (Shanghai, China) and were kept at 37 C in a 5% CO 2 humidifier incubator. The DMEM media supplemented with 10% FBS, 1% penicillin, and 1% streptomycin were used as the culture media.

| Cellular uptake
AGS cells were seeded onto cover-slide system at a density of

| Cellular cytotoxicity analysis
The antitumor activities of the micelles were evaluated on AGS cells using CCK-8 assays. Cells were seeded at a density of 5 Â 10 4 /ml in a 96-well plate for incubation. After overnight culturing, AGS cells were treated by PPH or PPH@5Fu at different concentrations. After 24 h further incubation, medium was removed and 20 μl CCK-8 solutions (5 mg/ml) were added. Cells were then incubated for 4 h at 37 C, the cytotoxicity of PPH or PPH@5Fu was analyzed by the CCK-8 assay. Likewise, we analyzed the cyotoxicity of PBS + NIR, PPH@5Fu, PPH@5Fu@ICG, PPH@ICG +-NIR, and PPH@5Fu@ICG + NIR by the same assay.

| Statistical analysis
All the quantitative results were presented as mean ± standard deviation. Students's t-test was utilized to evaluate the significance in two groups, and one-way ANOVA was used among multiple groups.
Herein, a probability value P < 0.05 (*) was accepted as statistically significant difference, and double asterisk (**) indicated P < 0.01, was considered as very significant difference. FTIR spectrometry was utilized to further confirm the synthesis of polymer. As shown in Figure S1B, the peak at 3680 cm À1 and 1740 cm À1 was the absorption band of the hydroxyl group and ester bond of PCL segments. The C-H symmetric stretching vibration peak of CH 3  which further resulted in lattice distortion, smaller lattice constants, and shortened bond lengths. As shown in Figure 1f, LCST was 43.4 C.
As shown in Figure S4, DSC curves of PPH@5Fu@ICG demonstrated LCST, indicating general agreement of both results.
ICG is a member of cyanine that generally consists of two heterocyclic units connected with linear alkene units. When irradiated by NIR laser, ICG molecules absorb luminous energy and reach to the high vibration energy level of excited state. Then, ICG molecules generate thermal energy and act out by hyperthermia and photothermal effects during the deactivation process. Therefore, the temperature increasing profiles under laser irradiation (808 nm, 1 W/cm 2 ) in vitro was monitored to evaluate the photothermal efficiency of PPH@ICG. As shown in Figure 2a,b, the temperature of free ICG and PPH@ICG aqueous solution showed a quick temperature increase. The maximal temperature change of free ICG (80 μg/ml) and PPH@ICG (80 μg/ml) at 5 min could reach to 55.5 and 64.9 C, respectively. The photothermal efficiency of ICG encapsulated in NPs was enhanced slightly, which was consistent with previous reports that ICG-containing micelles was more efficient to trigger laser-dependent temperature increase than free ICG. The thermo-sensitive micelles of the PPH@ICG (40 μg/ml) transformed from micellization to demicellization in the NIR lasertriggered high-temperature (up to 45.9 C) which was far above the LCST. This indicated that NIR laser stimulus was an effective tragedy to enhance drug release of PPH@ICG. In addition, the maximal temperature change of PBS only increased 1.7 C. As shown in The standard curve of 5Fu is shown in Figure 2e. In our study, the drug EE and loading efficiency (EF) of 5Fu in PPH@5Fu@ICG were 68.3% and 19.6%, respectively. The standard curve of ICG is shown in Figure S5. LCST), the original protective hydrophilic shell gradually precipitates from the aqueous solution due to phase transition, and then adsorbs on the hydrophobic core surface, resulting in the "core-shell" structure destruction of the micelle. In the process of phase transition, the water molecules in the micelle will also be squeezed out with the collapse of the hydrophilic shell. Meanwhile, some drug molecules will also be excreted, so that the release amount and release rate of thermo-sensitive polymer drug-loaded micelles at 45 C are significantly higher than those at 25 C.

| In vitro cellular uptake
Confocal laser scanning microscope was utilized to test the intracellular distribution of micelles and NIR laser-triggered endosomal disruption in cells. As shown in Figure 3a,  To further explore the cellular internalization process of ICG, flow cytometry analysis was performed. As shown in Figure 3b, ICG fluorescence signals of PPH@5Fu@ICG with NIR laser (808 nm, 1 W/cm 2 , 5 min) were largely internalized by AGS cells compared with that without NIR. Quantification of mean fluorescence intensity further revealed that the mean fluorescence intensity in AGS cells treated with NIR irradiation was significantly higher than that without NIR irradiation at 4 and 12 h ( Figure S6). To directly investigate the biocompatibility of the nanoparticles, the viability of AGS cells after being exposed to PPH or PPH@ICG with various concentrations were evaluated. As shown in Figure 4a, the results showed that cell viability was greater than 90% and even the PPH concentration was increased to 500 μg/ml, indicating that PPH and PPH@ICG had no obvious toxicity in AGS cells. Next, the cell viability of PBS, PPH@5Fu, PPH@5Fu@ICG, PPH@ICG + NIR, and PPH@5Fu@ICG + NIR at various 5Fu or ICG concentrations to AGS cells was further investigated, respectively. As shown in Figure 4b, PPH@5Fu showed a little lower cytotoxicity than PBS. However, PPH@5Fu@ICG + NIR caused a better effect to kill AGS cells than PPH@5Fu@ICG under higher 5Fu concentration (75 μg/ml) and ICG concentration (40 μg/ml), which was likely due to the synergistic effects of abrupt drug release and chemo/PTT. 36  The enhanced cytotoxic effects driven by NIR laser was further evaluated through cell apoptosis analysis. As shown in Figure 4c, the results showed that cell late apoptosis/necrosis was more efficiently induced by PPH@5Fu@ICG + NIR (32.0%) compared with cells treated with PPH@ICG + NIR (17.8%) and PBS (0.14%). The results evidently illustrated that NIR laser irradiation could obviously promote the effect of PPH@5Fu@ICG + NIR on apoptosis and cell death. 37 As shown in Figure 4d, TEM results of tumor cell apoptosis displayed cell shrinkage, membrane blebbing, and chromatin condensation.
Meanwhile, the PPH@5Fu@ICG nanoparticles with NIR irradiation group showed more black granular nanoparticles in AGS cells, which also implied much greater cellular uptake of the nanoparticle than without NIR irradiation.

| In vivo fluorescence imaging and distribution
In vivo fluorescence imaging was utilized to investigate the real-time distribution and intratumoral enrichment of PPH@5Fu@ICG micelles.
In the gastric tumor-bearing nude mice, the ICG fluorescence signals of PPH@5Fu@ICG group were much higher than pure ICG group at any time point (Figure 5a). ICG group was metabolized and cleared from blood circulation, and weak fluorescence was detected in tumor   Figure S7A showed the infrared thermographic maps of mice after 5 min laser irradiation. As shown in Figure S7B, the tumor temperature of PPH@5Fu@ICG group rose to 43.9 C after 5 min laser irradiation, which could initiate drug release ($43 C) and cause an irreversible damage to the tumor tissues. 39 However, the tumor temperature of ICG group can only reach to 38.3 C with same laser irradiation, which failed to cause irreversible damage to the tumor tissues. 40

| In vivo enhanced antitumor effect study
The antitumor effect of PPH@5Fu@ICG with 808 nm NIR irradiation  Table S1. These results indicated that the low dosage of 5Fu and NIR irradiation have a positive effect on the inhibition of tumors by chemotherapy and PTT. Compared with other groups, the tumor size of PPH@5Fu@ICG + NIR group was the smallest, which achieved the best antitumor effect. These results indicated that PPH@5Fu@ICG +-NIR group exhibited a significant improvement in antitumor efficacy due to the NIR-triggered thermo-chemotherapy synergy. 41,42 Moreover, the body weight of mice and H&E staining images of major organs were used to analyze treatment-induced toxic side effects. As shown in Figure 6c, 43 As shown in Figure 7d, HSP70 expression of PPH@5Fu@ICG + NIR group was significantly inhibited compared to PPH and PPH@5Fu@ICG groups, which indicated that the mechanism of NIR-induced photothermal chemotherapy is expression of HSP70.
Caspase-3 was a critical mediator of apoptosis, and enhanced caspase-3 indicated higher degrees of tumor cell apoptosis. 44 Ki67 could predict cancer progression as a marker of cell proliferation, and the high expression of Ki67 indicated more proliferating tumor cells. 45 Meanwhile, PPH@5Fu@ICG + NIR group showed over-expression of Caspase-3 and low-expression of Ki67 protein, which was due to the great inhibition of tumor growth and low side effect of NIR-induced photothermal chemotherapy. 46 The underlying mechanism of hyperthermia-induced cell death may be that up-regulate of HSP70 gene expression and inhibits caspase-3 activation and down-regulate of Ki67, resulting in mitochondrial instability yielding mitochondrial damage. 47 In summary, PPH@5Fu@ICG nanoparticles achieved an excellent tumor ablation effect with a low dose of 5-Fu and NIR irradiation in vivo, which could be traced by ICG NIR fluorescence imaging.

| CONCLUSION
In summary, we successfully developed a novel thermo-responsive 5-fluorouracil/ICG-coloaded micelles (PPH@5Fu@ICG) assembled by amphiphilic copolymers with excellent size distribution and high drug EF. Our results showed that PPH@5Fu@ICG exhibited excellent temperature response and NIR laser-controlled drug release. We evalu-  Zhen Zong: Funding acquisition (equal); resources (equal); writingreview and editing (equal).

CONFLICT OF INTEREST
There are no conflicts to declare.

DATA AVAILABILITY STATEMENT
Data available on request due to privacy/ethical restrictions.